Back to EveryPatent.com
| United States Patent |
5,274,141
|
|
Nicolaou
, et al.
|
December 28, 1993
|
Designed quinone- and hydroquinone-containing cyclic enediyneols and
enediyneones having DNA cleaving and cytotoxic properties
Abstract
Quinone- and hydroquinone-containing cyclic enediyneols and enediyneones
having 10-carbon atoms in the enediyne-containing ring that cleave DNA are
disclosed, as are methods of making and using the same.
| Inventors:
|
Nicolaou; Kyriacos C. (La Jolla, CA);
Liu; Aijun (Nutley, NJ)
|
| Assignee:
|
The Scripps Research Institute (La Jolla, CA)
|
| Appl. No.:
|
938756 |
| Filed:
|
September 1, 1992 |
| Current U.S. Class: |
552/220; 536/25.4; 552/298; 552/299 |
| Intern'l Class: |
C07C 050/06; C07C 050/14; C07C 050/20 |
| Field of Search: |
552/299,220,298
536/25.4
514/680,681,729,732
|
References Cited
U.S. Patent Documents
| 5136099 | Aug., 1992 | Skokotas et al. | 568/327.
|
| 5183942 | Feb., 1993 | Nicolaou et al. | 568/375.
|
Other References
Nicolaou et al., J. Am. Chem. Soc., 110:4866-4868 (1988).
|
Primary Examiner: Cintins; Marianne M.
Assistant Examiner: Kestler; Kimberly J.
Attorney, Agent or Firm: Dressler, Goldsmith, Shore & Milnamow, Ltd.
Goverment Interests
GOVERNMENTAL RIGHTS
This invention was made with support from the United States Government
under National Institutes of Health Grant CA 46446, and the United States
Government has certain rights in the invention.
Claims
We claim:
1. A compound of the formula
##STR20##
wherein .dbd.Y is .dbd.O or --OH and --H, and W together with the carbon
atoms of the depicted, intervening vinylene group forms a benzoquinoidal,
naphthoquinoidal or anthraquinoidal ring system or the corresponding
hydroquinoidal form thereof, with the proviso that when W forms a
corresponding hydroquinoidal ring system, .dbd.Y is --OH are --H.
2. The compound of claim 1 wherein W together with the carbon atoms of the
depicted, intervening vinylene group forms a benzoquinoidal or
hydroquinoidal ring system.
3. The compound of claim 1 wherein the benzoquinoidal, naphthoquinoidal or
anthraquinoidal ring system or corresponding hydroquinoidal form thereof
is substituted with a C.sub.1 -C.sub.6 alkyl or C.sub.1 -C.sub.6 alkoxy
group.
4. The compound of claim 1 wherein .dbd.Y is .dbd.O.
5. The compound of claim 1 wherein .dbd.Y is --OH and --H.
6. A compound selected from the group consisting of Formulas II and III,
below,
##STR21##
wherein .dbd.Y is .dbd.O or --OH and --H, and R.sup.1 and R.sup.2 are the
same or different and are selected from the group consisting of hydrogen,
C.sub.1 -C.sub.6 alkyl and C.sub.1 -C.sub.6 alkoxy.
7. The compound of claim 6 wherein R.sup.1 =R.sup.2.
8. The compound of claim 7 wherein R.sup.1 is a C.sub.1 -C.sub.6 alkyl
group.
9. The compound of claim 8 wherein said C.sub.1 -C.sub.6 alkyl group is
methyl.
10. A compound having a structural formula selected from the group
consisting of those shown below
##STR22##
11. A composition comprising a physiologically tolerable diluent having an
active agent dissolved or dispersed therein in a DNA cleaving amount, said
active agent being a compound of the formula
##STR23##
wherein .dbd.Y is .dbd.O or --OH and --H, and W together with the carbon
atoms of the depicted, intervening vinylene group forms a benzoquinoidal,
naphthoquinoidal or anthraquinoidal ring system or the corresponding
hydroquinoidal form thereof, with the proviso that when W forms a
corresponding hydroquinoidal ring system, .dbd.Y is --OH and --H.
12. The composition of claim 11 wherein the benzoquinoidal,
naphthoquinoidal or anthraquinoidal ring system or corresponding
hydroquinoidal form thereof is substituted with a C.sub.1 -C.sub.6 alkyl
or C.sub.1 -C.sub.6 alkoxy group.
13. The composition of claim 11 wherein W together with the carbon atoms of
the depicted, intervening vinylene group forms a benzoquinoidal or
hydroquinoidal ring system.
14. The composition of claim 13 wherein said active agent is a compound of
Formula II or III, below
##STR24##
wherein .dbd.Y is .dbd.O or --OH and --H, and R.sup.1 and R.sup.2 are the
same or different and are selected from the group consisting of hydrogen,
C.sub.1 -C.sub.6 alkyl and C.sub.1 -C.sub.6 alkoxy.
Description
DESCRIPTION
1. Technical Field
The present invention relates to novel DNA-cleaving and antitumor
compounds, and more specifically to a group of quinone- or
hydroquinone-containing cyclic enediyneols and enediyneones having ten
carbons in the enediyne ring.
2. Background Art
Natural products have been capturing the interest and imagination of
isolation, synthetic, and medicinal chemists for a very long time due to
their fascinating structures and biological activities. Man-designed
molecules ("designer molecules") with predefined chemical and biological
properties could enrich and complement this arsenal of substances, and
sharpen the capability of chemistry to deliver biologically and
therapeutically useful compounds.
Described herein are the design, synthesis, chemical and biological actions
of novel designer molecules with DNA cleaving and antitumor properties;
for some recent examples of designed DNA-cleaving molecules, see: (a)
Nicolaou et al., J. Am. Chem. Soc., 110:4866, 7247 (1988); (b) Nicolaou et
al., Angew. Chem. Int. Ed. Engl., 28:1272 (1989); (c) Povsic et al., J.
Am. Chem. Soc., 111:3059 (1989); (d) Hertzberg et al., J. Am. Chem. Soc.,
104:313 (1982); (e) Moser et al., Science, 238:645 (1987); (f) Corey et
al., J. Am. Chem. Soc., 111:8523 (1989); (g) Pyle et al., J. Am. Chem.
Soc., 111:4520 (1989); (h) Sigman, J. Am. Chem. Soc., 111:4941 (1989); (i)
Ohno et al., J. Am. Chem. Soc., 112:838 (1990); (j) Danishefsky, J. Org.
Chem., 54:2781 (1989); Nicolaou et al., Angew. Chem. Int. Ed. Engl.,
103:1032 (1991); and allowed U.S. patent application Ser. No. 07/788,161,
filed Nov. 5, 1991.
In addition to the man-made DNA cleaving compounds, naturally occurring
enediyne compounds have also been reported and studies. Included among the
naturally occurring enediynes are calicheamicin and esperamicin that have
substantially identical aglycon portions but different sugar portions [(a)
Lee et al., J. Am. Chem. Soc., 109:3464, 3466 (1987); (b) Nicolaou et al.,
J. Am. Chem. Soc., 110:7247 (1988); (c) Hawley et al., Proc. Natl. Acad.
Sci. USA, 86:1105 (1989); (d) Golik et al., J. Am. Chem. Soc., 109:3461,
3462 (1987)] and neocarzinostation that also contains sugar-derivative
side chains [(a) Edo et al., Tetrahedron Lett., 26:331 (1984); (b) Chin et
al. Biochemistry, 27:8106 (1988); (c) Lee et al., Biochemistry, 28:1019
(1989)].
BRIEF SUMMARY OF THE INVENTION
The invention contemplates novel DNA-cleaving, antibiotic and antitumor
compounds that contain a quinoid or hydroquinoid ring fused to a
10-membered enediyne ring that includes a hydroxyl or keto (oxo) group on
a carbon atom adjacent to a triple bond. General structural formulas for a
quinoid- or hydroquinoid-containing enediyneol compound and enediyneone
compound are shown below as structural Formula I.
##STR1##
wherein .dbd.Y is .dbd.O or --OH and --H, and
W together with the carbon atoms of the depicted, intervening vinylene
group forms a benzoquinoidal, naphthoquinoidal or anthraquinoidal ring
system or the corresponding hydroquinoidal form thereof, with the proviso
that when W forms a corresponding hydroquinoidal ring system, .dbd.Y is
--OH and --H.
A preferred group of compounds of structural Formula I are the compounds of
structural Formulas II and III, below,
##STR2##
wherein .dbd.Y is .dbd.O or --OH and --H, and
R.sup.1 and R.sup.2 are the same or different and are selected from the
group consisting of hydrogen, C.sub.1 -C.sub.6 alkyl and C.sub.1 -C.sub.6
alkoxy.
It is preferred that R.sup.1 and R.sup.2 be the same and C.sub.1 -C.sub.6
alkyl. A compound wherein R.sup.1 and R.sup.2 are both methyl is most
preferred.
A pharmaceutical composition that contains a before-defined compound
present in a DNA-cleaving, or tumor growth-inhibiting amount dissolved or
dispersed in a physiologically tolerable diluent is also contemplated.
A method utilizing a before-discussed composition containing a compound of
structural Formula I is also contemplated. Here, DNA to be cleaved or
target tumor cells whose growth is to be inhibited are contacted with a
compound of Formula I, preferably in a before-described composition. That
contact is maintained for a time period sufficient for the desired result
to be effected. Multiple administrations of the composition are also
contemplated.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings forming a portion of this disclosure,
FIG. 1 is a photograph of an ethidium bromide stained 1 percent agarose
electrophoresis gel o that illustrates the cleavage of .phi.X174 form I
DNA (100 .mu.M base pairs) by Compounds 7, 8, 9, 10 and 12 at 50-5000
.mu.M after 15 hours at 37.degree. C. at a pH value of 7.4 in 50 .mu.M
Tris HCl buffer. Lane 1 shows the DNA alone as control. Lanes 2-10
correspond to the following: Lane 2: Compound 12 (100 .mu.M); Lane 3:
Compound 10 (100 .mu.M); Lane 4: Compound 7 (100 .mu.M); Lane 5: Compound
8 (100 .mu.M); Lanes 6-10: Compound 9 (50, 100, 500, 1000 and 5000 .mu.M).
Forms I, II and III shown to the left of the photograph show the relative
migrations of supercoiled (form I) relaxed (form II) and linear (form III)
DNA, respectively.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present invention relates to compounds, compositions and methods for
cleaving DNA and thereby killing targeted cells such as tumor cells. Three
related general types of compounds are contemplated.
The first is a quinoidal enediyneol that contains ten carbon atoms in the
enediyneol ring such as Compound 8. These compounds can themselves cleave
DNA and are cytotoxic to cancer cells such as Molt-4 leukemia cells.
The second is a hydroquinoidal enediyneol that can be prepared from an
above quinoidal enediyneol and also contains ten carbon atoms in the
enediyneol ring such as Compound 9. These compounds also cleave DNA, but
are not particularly cytotoxic. However, the hydroquinone is readily
oxidized in vivo. These compounds can therefore be viewed as prodrugs for
a quinoidal enediyneol compound.
The third compound type is a quinoidal enediyneone. This type of compound
can be prepared from a quinoidal enediyneol, cleaves DNA and is cytotoxic
to cancer cells such as Molt-4 cells.
II. The Compounds
A compound contemplated by the present invention corresponds to structural
Formula I, below,
##STR3##
wherein .dbd.Y is .dbd.O or --OH and --H, and
W together with the carbon atoms of the depicted, intervening vinylene
group forms a benzoquinoidal, naphthoquinoidal or anthraquinoidal ring
system or the corresponding hydroquinoidal form thereof, with the proviso
that when W forms a corresponding hydroquinoidal ring system, --Y is --OH
and --H.
A preferred group of compounds of structural Formula I are the compounds of
structural Formulas II and III, below,
##STR4##
wherein .dbd.Y is .dbd.O or --OH and --H, and
R.sup.1 and R.sup.2 are the same or different and are
selected from the group consisting of hydrogen, C.sub.1 -C.sub.6 alkyl and
C.sub.1 -C.sub.6 alkoxy. Here, W of structural Formula I formed a
hydrobenzoquinone or benzoquinone, respectively.
A preference as to R.sup.1 and R.sup.2 is that both be the same group;
i.e., if R.sup.1 is C.sub.1 -C.sub.6 alkyl, R.sup.2 is C.sub.1 -C.sub.6
alkyl. The basis for this preference is ease of chemical synthesis in that
if R.sup.1 and R.sup.2 are different from each other, mixtures of isomers
can be formed that could require separation and loss of overall yield,
whereas if R.sup.1 =R.sup.2 there would be no mixtures.
The hydroxyl of the 10-membered ring of Formula I or that of the other
formulas herein, where .dbd.Y is --OH and --H, can be above (.beta.) or
below (.alpha.) the plane of that ring as shown, as can the hydrogen atom
that is not shown. Those .alpha. and .beta. configurations are indicated
by a wavy line in Formula II and formulas elsewhere herein. In addition,
where a hydrogen is not needed to show stereochemistry or to complete a
group such as a methylene or a hydroxyl group, that hydrogen is not shown
following usual conventions.
Exemplary compounds, in addition to the benzoquinoids shown before, wherein
W together with the carbon atoms of the depicted, intervening vinylene
group forms naphthoquinoidal or anthraquinoidal ring systems or their
corresponding hydroquinoidal forms are illustrated below by structural
Formulas IV-IX, wherein .dbd.Y is as before-defined.
##STR5##
The W quinoidal or hydroquinoidal ring system can have bonded to it, at
various positions (other than those required for the fusion to the
depicted vinylene carbon atoms) hydrogen or a variety of substituents.
Exemplary substituents include methyl, ethyl, isopropyl, n-propyl,
isobutyl, sec-butyl, t-butyl, hexyl, and cyclohexyl (C.sub.1 -C.sub.6
alkyl) and methoxy, ethoxy, propoxy, butoxy, iso-butoxy, cyclopentyloxy,
and cyclohexyloxy (C.sub.1 -C.sub.6 alkoxy).
For ease of synthesis, because of the formation of isomeric products, it is
preferred that a compound of Formula I include a plane of symmetry that
can be drawn bisecting the vinylene bond and any quinoid of hydroquinoid
ring system W bonded thereto, with the .alpha. or .beta. stereochemistry
of the hydroxyl group being neglected. Similarly, any substituents present
on a quinoid or hydroquinoid ring system W are preferably symmetrically
substituted about the depicted vinylene bond of Formula I.
Of the above W ring systems, a benzo ring is preferred. A substituted benzo
ring is also contemplated, as noted for a compound of Formulas II and III,
as are other substituted ring systems W, and substituents can be bonded at
the remaining positions not utilized in the fusion to the depicted
vinylene carbons. Structural Formulas X-XV for further exemplary compounds
are illustrated below, wherein .dbd.Y and R.sup.1 and R.sup.2 are as
previously defined.
##STR6##
Reference is made herein to the carbon atoms of the vinylene group of
depicted structural formulas. Following modern theories, those carbon
atoms from sp.sup.2 bonds. Atoms in the quinoidal and hydroquinoidal W
groups also form sp.sup.2 bonds.
Thus, when W together with the intervening carbon atoms of a depicted
vinylene group form a quinoidal or hydroquinoidal ring system the bonding
of those carbon atoms of the depicted vinylene group remains sp.sup.2.
Nevertheless, usual structural formulas for quinoidal or hydroquinoidal
compounds that utilize alternating double and single bonds can show a
single bond rather than a double bond at the position occupied by a
depicted vinylene group.
In view of the fact that the hybridization of the carbons of a depicted
vinylene group remain sp.sup.2, and such sp.sup.2 hybridization rather
than the presence of a vinylene group, per se, is what is of importance in
the reactions of a compound of the invention, the absence of a
"formalized", double bond in a written formula for a compound of Formula I
is of no consequence.
In reviewing the above preferences it is seen that in particularly
preferred practice, R.sup.1 =R.sup.2 In addition, particularly preferred
R.sup.1 and R.sup.2 groups are methyl. Structural formulas for the
particularly preferred hydroquinoidal enediyneol of Formula II (Compound
9), the quinoidal enediyneol and quinoidal enediyneone of Formula III
(Compounds 8 and 10, respectively), are shown below.
##STR7##
III. Syntheses
A compound of the invention is prepared readily. A detailed description of
the preparation of exemplary benzoquinoid and hydroquinoid, preferred
Compounds 8, 9 and 10 is provided in Scheme 1 and the adjoining text,
hereinafter. The discussion that follows will therefore center on
preparation of compounds where W together with the depicted vinylene group
carbon atoms forms a naphthoquinoidal or anthraquinoidal ring system.
In a preferred method of synthesis, the portions of the molecule containing
the triple bonds are added separately to a vicinyl-dihalohydroquinone
portion as silyl-containing entities. The silyl groups of the resulting
enediyne compound are then removed, and formed into a halide and an
aldehyde, and are then linked to form a 10-membered hydroxyl-containing
cyclic enediyne.
As noted earlier, it is preferred that the whole molecule be symmetrical
with a plane of symmetry bisecting the vinylene group. In view of the fact
that difficultly separable isomers can arise when R.sup.1 and R.sup.2 are
not identical, it is also preferred that R.sup.1 and R.sup.2 be identical
as noted before.
Notwithstanding the above preferences for a symmetrical compound and
sameness of R.sup.1 and R.sup.2, asymmetric molecules are also
contemplated. Such molecules are, however, less preferred because of the
difficulties involved with separation of the various isomers that can be
produced.
One starting material is a vicinyldihalohydroquinone. Such compounds are
well known in the art, and can be prepared from their usually more
available corresponding quinone forms by gentle reduction. For example,
2,3-dichloro-1,4-naphthoquinone, available from Aldrich Chemical Co.,
Inc., Milwaukee, Wis., can be reduced with a borohydride reagent or
aqueous dithionite ion to form the corresponding hydroquinone. The vicinyl
dihalide can be prepared from a quinone as is illustrated hereinafter or
from a hydroquinone.
It is preferred that the vicinyl halo groups of the quinone or hydroquinone
be the same halogen. That halogen can be a chloro, bromo, or iodo group,
with iodo groups being preferred. Preferred vicinyl iodo compounds can be
prepared from corresponding dichloro or dibromo compounds by an iodine
exchange reaction.
Suitable C.sub.1 -C.sub.6 alkylated vicinyl dihaloquinones and
hydroquinones are also known in the art. Suitable C.sub.1 -C.sub.6
dialkoxy vicinyl dihalohydroquinones can be prepared by C.sub.1 -C.sub.6
alkylation of the corresponding quinonediols, followed by reduction of the
oxo groups and then halogenation. For example, quinizarin
(1,4-dihydroxyanthraquinone), also available from Aldrich, can be
alkylated as with dimethyl sulfate, reduced as discussed before and then
dihalogenated under standard conditions for aromatic compounds.
A suitable vicinyl dihalohydroquinone is then sequentially reacted and
condensed with HC.ident.CSiMe.sub.3 (Me.dbd.CH.sub.3) and then with
HC.ident.C(CH.sub.2).sub.4 OSi.sup.t BuMe.sub.2 (.sup.t Bu.dbd.t-butyl) to
form a corresponding open chain enediyne whose triple bond-containing
groups are protected with the above silyl groups. The above condensations
are carried out by use of a Pd(O)-Cu(I) catalyst in a solvent such as
benzene and in the presence of an excess of an amine such as
di-isopropylamine.
The hydroquinone hydroxyls are then protected with a base insensitive
protecting group other than a silyl group, with pivaloyl groups being
preferred. A bis(pivaloyl) ester is thus formed.
The silyl protecting groups are then separately removed to form the
respective acetylene- and hydroxyl termini. The acetylene hydrogen atom is
replaced with a halogen, preferably an iodo group, and the hydroxyl group
is gently oxidized to form an aldehyde, as with pyridinium chlorochromate
(PCC). The 10-membered ring is then closed in the presence of an excess of
CrCl.sub.2 and a catalytic amount of NiCl.sub.2 to form the
hydroxyl-containing 10-membered ring enediyne.
Removal of the pivaloyl groups as with excess LiAlH.sub.4 provides a
quinone of structural Formula I, where .dbd.Y is --OH and --H. The quinone
form is recovered via a presumed air-sensitive corresponding hydroquinone
precursor. Reaction of the quinone with a mild oxidant such as PCC in the
presence of 4.ANG. molecular sieves provides a compound of structural
Formula I where .dbd.Y is .dbd.O. On the other hand, reaction of that
quinone with a reducing agent such as dithionite ion provides the
hydroquinone form of a compound of structural Formula I where .dbd.Y is
--OH and --H.
IV. Pharmaceutical Compositions
A compound of structural Formula I is useful as a DNA cleaving agent, as
are dynemicin A, calicheamicin, esperamicin and neocarzinostatin. A
compound of the invention can also therefore be referred to as an "active
agent" or "active ingredient". A compound of that structural formula can
also be used to inhibit the growth of neoplastic cells as can those known
compounds in that cytotoxicity toward tumor cells by those previously
known compounds proceeds via DNA cleavage, at least in part. Thus, a
pharmaceutical composition of a compound of structural Formula I is
contemplated.
Cleavage of cell-free DNA can be assayed using the techniques described
hereinafter as well as those described by Mantlo et al., J. Org. Chem.,
54:2781 (1989); Nicolaou et al., J. Am. Chem. Soc., 110:7147 (1989);
Nicolaou et al., J. Am. Chem. Soc., 110:7247 (1988) or Zein et al.,
Science, 240:1198 (1988) and the citations therein.
A before-described compound can also be shown to undergo a Bergman
cycloaromatization reaction in the presence of benzyl mercaptan,
1,4-cyclohexadiene or other hydrogen donor as discussed in Haseltine et
al., J. Am. Chem. Soc., 111:7638 (1989). This reaction forms a
benzene-containing reaction product as is formed during DNA cleavage, and
can be used as a co-screen to select more active compounds.
A pharmaceutical composition is thus contemplated that contains a
before-described compound of the invention as active agent. A
pharmaceutical composition is prepared by any of the methods well known in
the art of pharmacy all of which involve bringing into association the
active compound and the carrier therefor. For therapeutic use, a compound
utilized in the present invention can be administered in the form of
conventional pharmaceutical compositions. Such compositions can be
formulated so as to be suitable for oral or parenteral administration, or
as suppositories. In these compositions, the agent is typically dissolved
or dispersed in a physiologically tolerable carrier or diluent.
A carrier or diluent is a material useful for administering the active
compound and must be "pharmaceutically acceptable" in the sense of being
compatible with the other ingredients of the composition and not
deleterious to the recipient thereof. Thus, as used herein, the phrases
"physiologically tolerable" or "pharmaceutically acceptable" are used
interchangeably and refer to molecular entities and compositions that do
not produce an allergic or similar untoward reaction, such as gastric
upset, dizziness and the like, when administered to a mammal. The
physiologically tolerable carrier can take a wide variety of forms
depending upon the preparation desired for administration and the intended
route of administration.
As an example of a useful composition, a compound of the invention (active
agent) can be utilized, dissolved or dispersed in a liquid composition
such as a sterile suspension or solution, or as isotonic preparation
containing suitable preservatives. Particularly well-suited for the
present purposes are injectable media constituted by aqueous injectable
buffered or unbuffered isotonic and sterile saline or glucose solutions,
as well as water alone, or an aqueous ethanol solution. Additional liquid
forms in which these compounds can be incorporated for administration
include flavored emulsions with edible oils such as cottonseed oil, sesame
oil, coconut oil, peanut oil, and the like, as well as elixirs and similar
pharmaceutical vehicles. Exemplary further liquid diluents can be found in
Remminqton's Pharmaceutical Sciences, Hack Publishing Co., Easton, Pa.
(1980).
An active agent can also be administered in the form of liposomes. As is
known in the art, liposomes are generally derived from phospholipids or
other lipid substances. Liposomes are formed by monoor multi-lamellar
hydrated liquid crystals that are dispersed in an aqueous medium. Any
non-toxic, pharmaceutically acceptable and metabolizable lipid capable of
forming liposomes can be used. The present compositions in liposome form
can contain stabilizers, preservatives, excipients, and the like in
addition to the agent. The preferred lipids are the phospholipids and the
phosphatidyl cholines (lecithins), both natural and synthetic.
Methods of forming liposomes are known in the art. See, for example,
Prescott, Ed., Methods in cell Biology, Vol. XIV, Academic press, New
York, N.Y. (1976), p.33 et seq.
An active agent can also be used in compositions such as tablets or pills,
preferably containing a unit dose of the compound. To this end, the agent
(active ingredient) is mixed with conventional tableting ingredients such
as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium
stearate, dicalcium phosphate, gums, or similar materials as non-toxic,
physiologically tolerable carriers. The tablets or pills can be laminated
or otherwise compounded to provide unit dosage forms affording prolonged
or delayed action.
It should be understood that in addition to the aforementioned carrier
ingredients the pharmaceutical composition described herein can include,
as appropriate, one or more additional carrier ingredients such as
diluents, buffers, flavoring agents, binders, surface active agents,
thickeners, lubricants, preservatives (including antioxidants) and the
like, and substances included for the purpose of rendering the formulation
isotonic with the blood of the intended recipient.
The tablets or pills can also be provided with an enteric layer in the form
of an envelope that serves to resist disintegration in the stomach and
permits the active ingredient to pass intact into the duodenum or to be
delayed in release. A variety of materials can be used for such enteric
layers or coatings, including polymeric acids or mixtures of such acids
with such materials as shellac, shellac and cetyl alcohol, cellulose
acetate phthalate, and the like. A particularly suitable enteric coating
comprises a styrene-maleic acid copolymer together with known materials
that contribute to the enteric properties of the coating. Methods for
producing enteric coated tablets are described in U.S. Pat. No. 4,079,125
to Sipos, which is herein incorporated by reference.
The term "unit dose", as used herein, refers to physically discrete units
suitable as unitary dosages for administration to warm blooded animals,
each such unit containing a predetermined quantity of the agent calculated
to produce the desired therapeutic effect in association with the
pharmaceutically acceptable diluent. Examples of suitable unit dosage
forms in accord with this invention are tablets, capsules, pills, powder
packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules,
vials, segregated multiples of any of the foregoing, and the like.
A compound of the invention is present in such a pharmaceutical composition
in an amount effective to achieve the desired result. For example, where
in vitro cell-free DNA cleavage is the desired result, a compound of the
invention can be utilized in an amount sufficient to provide a
concentration of about 1.0 to about 10,000 micromolar (.mu.M) with a DNA
concentration of about 0.02 .mu.g/mL. As a cytotoxic (antitumor) agent, an
effective amount of a compound of the invention is about 0.1 to about 50
mg per kilogram of body weight or an amount sufficient to provide a
concentration of about 0.01 to about 100 .mu.g/mL to the bloodstream. In
in vitro cytotoxicity assays, a compound of structural Formula I exhibits
IC.sub.50 values on the order of 10.sup.-4 -10.sup.-7 M, depending upon
the cells assayed and compound utilized. Inasmuch as all of the above uses
ultimately result from DNA cleavage, the above amounts of an active agent
are referred to as DNA cleaving amounts.
V. Methods
A contemplated compound is useful in cleaving cell-free DNA, and also in
inhibiting the growth (killing) of neoplastic tumor cells via DNA
cleavage, and is utilized in a method for effecting such a result. Such a
compound is typically utilized in a before-described composition.
In accordance with such a method, cell-free DNA to be cleaved or target
neoplastic tumor cells to be killed are contacted in an aqueous medium
with a compound of Formula I, preferably in a before-described composition
that contains a contemplated compound of the invention (active ingredient)
present in an amount effective or sufficient for such a purpose; i.e., in
a DNA cleaving amount, dissolved or dispersed in a physiologically
tolerable (pharmaceutically acceptable) diluent. That contact is
maintained for a time sufficient for the desired result to be obtained;
i.e., cell-free DNA cleaved, or neoplastic cell growth inhibited.
Where the desired result is carried out in vitro contact is maintained by
simply admixing the cell-free DNA or target cells with the compound in an
aqueous medium and maintaining them together under the appropriate
conditions of temperature, pH, and the presence of nutrients for cell
growth to occur; i.e., culture conditions, as for control, untreated
cells. Thus, a single admixing and contacting is typically sufficient for
in vitro purposes, whether the DNA is in cell-free form or within living
cells.
The above method is also useful in vivo, as where a mammal such as a rodent
like a rat, mouse, or rabbit, a farm animal like a horse, cow or goat, or
a primate like a monkey, ape or human is treated. Here, contact of a
compound and the cells to be killed via DNA cleavage is achieved by
administration of the composition to the mammal by oral, nasal or anal
administration or by introduction intravenously, subcutaneously or
intraperitoneally. Thus, contact in vivo is achieved via the blood or
lymph systems as the aqueous medium.
Although a single administration (admixture) and its resulting contact is
usually sufficient to maintain the required contact and obtain a desired
result in vitro, multiple administrations are typically utilized in vivo.
Thus, because of a body's breakdown and excreting pathways, contact
between an active agent and the target cells is typically maintained by
repeated administration of a compound of the invention over a period of
time such as days, weeks or months, or more, depending upon the target
cells.
Thus, an aqueous medium can be a buffer system for cleaving cell-free DNA,
a nutrient-containing culture medium as for cytotoxicity studies or a body
fluid such as blood or lymph.
The time, temperature and pH value for maintenance can also be varied. For
cleaving cell-free DNA, 0.5-2 days at a temperature of about
25.degree.-50.degree. C. and pH value of about 7-9 can be used. For DNA
cleavage in cytotoxicity studies, tissue culture temperatures and pH
values as are well known are used with maintenance times of about 0.5-4
days. For in vivo use, the host animal's body temperature, body fluid pH
values and metabolism/catabolism/excretion control maintenance parameters.
Exemplary methods of the invention for cell-free DNA cleavage and in vitro
cytotoxicity of cancer cell lines and normal cells are illustrated
hereinafter.
VI. Results
Exemplary compounds 8-10 (shown previously) were targeted for synthesis for
their novel structures and potential chemical and biological properties.
Compounds 8-10 were prepared by the route shown below in Scheme 1. (It is
noted that the R and subscripted R groups used in the scheme that follows
have the meanings shown in the scheme as compared to the prior definitions
for superscripted R groups.)
##STR8##
Thus, the readily available diiodide Compound 1 was sequentially coupled
With Me.sub.3 SiC.ident.CH and .sup.t BuMe.sub.2 SiO(CH.sub.2).sub.4
C.ident.CH under the catalytic influence of Pd(O)--Cu(I) to afford the
diacetylenic Compound 3 via Compound 2 Diiodide Compound 1 was readily
prepared from 1,3-dimethylhydroquinone in 82 percent overall yield by
bromination followed by iodine exchange. 3.1 Equivalents of
HC.ident.CSiMe.sub.3, 0.66 equivalents of (PH.sub.3 P).sub.2 PdCl.sub.2,
0.17 equivalents of CuI and 2.0 equivalents of .sup.i Pr.sub.2 NH were
admixed and reacted with Compound 1 in benzene at 25.degree. C. for 8
hours in step a to form Compound 2 in 58 percent yield. Compound 2 was
then reacted in step b with 6 equivalents of HC.ident.C(CH.sub.2).sub.4
OSi.sup.t BuMe.sub.2, 0.1 equivalents of (Ph.sub.3 P).sub.4 Pd, 0.16
equivalents of CuI and 6.5 equivalents of .sup.i Pr.sub.2 NH in benzene at
25.degree. C. for 13 hours to form Compound 3 in 88 percent yield.
Formation of the bis(pivaloyl) ester of Compound 3 followed by desilylation
furnished Compound 4 in 71 percent overall yield as step c. Thus, 10
equivalents of .sup.t BuCOCl in pyridine were reacted with Compound 3 at
60.degree.-65.degree. C. for one hour to quantitatively produce the
bis(pivaloyl) ester. That bis ester was then placed in excess
BF.sub.3.OEt.sub.2 in CH.sub.2 Cl.sub.2 at 35.degree. C. for 4.5 hours to
remove the SiMe.sub.3 group. The resulting product was then reacted with
tetrabutylammonium fluoride (TBAF) in THF at 25.degree. C. for five
minutes to provide Compound 4 in a 71 percent yield for the final two
steps.
Iodination of the terminal acetylene of Compound 4 with iodine-morpholine
led to iodide Compound 5 which served as a precursor to iodoaldehyde
Compound 6. Thus, Compound 4 was reacted in step d with 5.0 equivalents of
I.sub.2 and excess morpholine in benzene at 45.degree. C. for 40 minutes
to provide Compound 5 in 89 percent yield. Compound 6 was prepared in 86
percent yield from Compound 5 in step e by reaction with 2.5 equivalents
of PCC in CH.sub.2 Cl.sub.2 at 25.degree. C. for 1.5 hours.
The crucial ring closure of Compound 6 was carried out with CrCl.sub.2
-NiCl.sub.2 system [Takai et al., J. Am Chem. Soc., 108:6048 (1986); and
Crevisy et al., Tetrahedron Lett., 32:3171 (1991)] leading to the targeted
protected enediyneol Compound 7 in step f. Here, Compound 6 was reacted
with 9.0 equivalents of CrCl.sub.2 and 0.64 equivalents of NiCl.sub.2 in
THF at 25.degree. C. for 4.5 hours to provide compound 7 in 75 percent
yield.
Compound 7 proved to be quite stable at ambient temperature and was
smoothly deprotected in step g by exposure to excess LiAlH.sub.4 (in THF
at -78.degree..fwdarw.zero degrees C for ten minutes) to afford, after
workup, the quinone Compound 8 in 96 percent yield, presumably through the
intermediacy of the air-sensitive hydroquinone Compound 9. Reduction of
the thermally labile Compound 8 to the hydroquinone Compound 9 in step h,
relatively stable under neutral conditions, was smoothly effected by
treatment with saturated aqueous Na.sub.2 S.sub.2 O.sub.4 (91 percent
yield). Finally, PCC oxidation (4.0 equivalents PCC, 4.ANG. molecular
sieves in CH.sub.2 Cl.sub.2 at 25.degree. C. for 0.5 hour) of Compound 8
afforded the tricarbonyl Compound 10 in 49 percent yield as step i.
Compound 10 proved to be the most reactive member of the series 7-10 in
cycloaromatization studies. Compound 8 was the most potent in cytotoxicity
studies.
Cycloaromatization studies with Compound 7-10 using 1,4-cyclohexadiene in
toluene revealed the following half lifes: Compound 7 (t1/2=74 hours at
110.degree. C.); Compound 8 (t1/2=2.6 hours at 55.degree. C.); Compound 9
(110.degree. C., complicated kinetics due to ease of conversion to
Compound 8 even under oxygen extrusion conditions, but seems quite stable
prior to oxidation); Compound 10 (t1/2=32 minutes at 55.degree. C.). These
reactions provided Compounds 11-13 as shown schematically in Scheme 2,
below.
##STR9##
The interaction of Compounds 7-10 and 12 with cell-free supercoiled DNA
(.phi.X174) at pH 7.4 and 37.degree. C. is shown in FIG. 1. The stable
pivalate derivative Compound 7 exhibited no DNA cleaving activity, whereas
Compounds 8, 9 and 10 showed significant DNA damaging properties. It is
presumed that, the damage on DNA caused by these agents is at least partly
due to their abilities to produce diradical species. Compound 12 exhibited
minor activity.
Assayed against a variety of cell lines, these compounds exhibited varying
degree of antitumor activity with the most impressive results obtained
with Compounds 8 and 10 [e.g. Molt-4 leukemia cells, IC.sub.50 for
Compound 8: 5.0.times.10.sup.-7 M; IC.sub.50 for Compound 10:
1.times.10.sup.-6 M]. These studies are discussed in greater detail
hereinafter.
EXAMPLE 1
Compound 3
##STR10##
R.sub.f : 0.37 (petroleum ether:EtOAc=14:1); 88 percent yield; IR
(CCl.sub.4) .nu..sub.max 3512, 2954, 2857, 2143 cm.sup.-1 ; .sup.1 H NMR
(500 MHz, CDCl.sub.3) .delta.5.61, 5.59 (2s, 2H, phenolic OH), 3.67 (t,
J=6.6 Hz, 2H, CH.sub.2 O), 2.56 (t, J=6.6 Hz, 2H, C.ident.CCH.sub.2),
2.18, 2.17 (2s, 6H, 2 aromatic CH.sub.3), 1.73-1.71 (m, 4H, CH.sub.2
CH.sub.2), 0.90 (s, 9H, C(CH.sub.3).sub.3), 0.28 (s, 9H,
Si(CH.sub.3).sub.3), 0.06 (s, 6H, 2.times.CH.sub.3 of TBDMS); .sup.13 C
NMR (125 MHz, CDCl.sub.3) .delta. 149, 148, 126, 124, 118, 117, 114, 100,
99, 74, 63, 32, 26, 25, 20, 13, 12, 0, -5; HRMS (FAB) calcd for C.sub.25
H.sub.44 O.sub.3 Si.sub.2, 444.2516, found 444.2525.
EXAMPLE 2
Compound 4
##STR11##
R.sub.f : 0.20 (petroleum ether:EtOAc=2:1); 71 percent yield; mp:
119.degree.-120.degree. C.; IR (CCl.sub.4) .nu..sub.max 3543, 2972, 2871,
1754 cm.sup.-1 ; .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 3.66 (t, J=6.5
Hz, 2H, CH.sub.2 OH), 3.34 (s, 1H, C.ident.CH), 2.45 (t, J=6.5 Hz, 2H,
C.ident.CCH.sub.2), 2.05 (2s, 6H, 2 aromatic CH.sub.3), 1.77-1.62 (m, 4H,
CH.sub.2 CH.sub.2), 1.40 (s, 18H, 2.times.C(CH.sub.3).sub.3); .sup.13 C
NMR (125 MHz, CDCl.sub.3) .delta. 176, 149, 147, 132, 130, 120, 118, 98,
88, 75, 72, 63, 39, 32, 27, 25, 19, 13; HRMS (FAB) calcd for C.sub.26
H.sub.34 O.sub.5 Cs (M+Cs) 559.1461, found 559.1491; Anal. calcd for
C.sub.26 H.sub.36 O.sub.5 : C, 73.21; H, 8.03; found C, 73.11; H, 8.09.
EXAMPLE 3
Compound 6
##STR12##
R.sub.f : 0.18 (petroleum ether:EtOAc=4:1); 86 percent yield; mp:
126.degree.-127.degree. C.; IR (CCl.sub.4) .nu..sub.max 2959, 2872, 1757,
1729 cm.sup.-1 ; .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 9.87 (s, 1H,
CHO), 2.75 (t, J=6.5 Hz, 2H, CH.sub.2 CHO), 2.50 (t, J=6.5 Hz, 2H,
C.ident.CCH.sub.2), 2.05, 2.04 (2s, 6H, 2 aromatic CH.sub.3), 1.90 (dddd,
J=6.5 Hz, 2H, --CH.sub.2 --), 1.40, 1.39 (2s, 18H,
2.times.C(CH.sub.3).sub.3); .sup.13 C NMR (125 MHz, CDCl.sub.3) .delta.
202, 176, 149, 147, 132, 131, 120, 119, 97, 89, 85, 76, 43, 39, 27, 21,
19, 13; HRMS (FAB) calcd for C.sub.26 H.sub.31 IO.sub.5 Cs (M+Cs)
683.0271, found 683.0271.
EXAMPLE 4
Compound 7
##STR13##
R.sub.f : 0.19 (petroleum ether:EtOAc=4:1); 75 percent yield; mp:
185.degree. C. (decomp.); UV (CHCl.sub.3) .lambda..sub.max (relative
intensity) 242 (1.59), 269 (0.78), 314 (0.18) nm; IR (CCl.sub.4)
.nu..sub.max 3400, 2973, 2872, 1755 cm.sup.-1 ; .sup.1 H NMR (500 MHz,
CDCl.sub.3) .delta. 4.55 (dd, J=8.9, 4.2 Hz, 1H, CHOH), (2.44-2.42 (m, 2H,
C.ident.CCH.sub.2), 2.17-2.10 (m, 2H, CH.sub.2 CHOH), 2.10 (brs, 6H, 2
aromatic CH.sub.3), 1.42, 1.41 (2s, 18H, 2.times.C(CH.sub.3).sub.3);
.sup.13 C NMR (125 MHz, CDCl.sub.3) .delta. 176, 146, 145, 131, 130, 122,
121, 104, 102, 81, 78, 63, 39, 38, 27, 24, 21, 13; HRMS (FAB) calcd for
C.sub.26 H.sub.32 O.sub.5 Cs (M+Cs) 557.1304, found 557.1315; Anal. calcd
for C.sub.26 H.sub.32 O.sub.5 : C, 73.56; H, 7.06; found C, 73.55; H,
7.58.
EXAMPLE 5
Compound 8
##STR14##
R.sub.f : 0.40 (petroleum ether:EtOAc=4:1); 96 percent yield; mp:
80.degree. C. (decomp.); UV (CHCl.sub.3) .lambda..sub.max (relative
intensity) 238 (0.61), 260 (0.64), 298 (1.18), 423 (0.12) nm; IR
(CCl.sub.4) .nu..sub.max 3290, 2935, 2197, 1654 cm.sup.-1 ; .sup.1 H NMR
(500 MHz, CDCl.sub.3) .delta. 4.69 (dd, J=8.5, 3.0 Hz, 1H, CHOH), 2.50
(ABqdd, Du=31.3 Hz, J=15.0, 8.5, 2.5 Hz, 2H, C.ident.CCHH.sub.2),
2.27-2.08 (m, 3H, CH.sub.2 CHOH and one of --CH.sub.2 --), 2.04 (s, 6H, 2
aromatic CH.sub.3), 1.88-1.79 (m, 1H, one of --CH.sub.2 --); .sup.13 C NMR
(125 MHz, CDCl.sub.3) .delta. 182, 141, 138, 137, 129, 116, 111, 81, 79,
63, 37, 23, 22, 13; HRMS (FAB) calcd for C.sub.16 H.sub.14 O.sub.3 Cs
(M+Cs) 386.9997, found 387.0005.
EXAMPLE 6
Compound 9
##STR15##
R.sub.f : 0.25 (petroleum ether:EtOAc=1:1); 91 percent yield; mp:
185.degree. C. (decomp.); UV (CHCl.sub.3) .lambda..sub.max (relative
intensity) 241 (0.83), 285 (0.32), 347 (0.45) nm; IR (CCl.sub.4)
.nu..sub.max 3042, 2978, 2876 cm.sup.-1 ; .sup.1 H NMR (500 MHz,
acetone-d.sub.6) .delta. 7.43, 7.21 (2s, 2H, phenolic OH), 4.53 (ddd,
J=8.5, 6.0, 3.0 Hz, 1H, CHOH), 4.29 (d, J=6.0 Hz, 1H, OH), 2.43-2.40 (m,
2H, C.ident.CCH.sub.2), 2.13 (s, 6H, 2 aromatic CH.sub.3), 2.13-2.05 (m,
3H, CH.sub.2 CHOH and one of --CH.sub.2 --), 1.75-1.67 (m, 1H, one of
--CH.sub.2 --); .sup.13 C NMR (125 MHz, THF-d.sub.8) .delta. 149, 148,
126, 125, 114, 113, 104, 103, 82, 80, 64, 40, 25, 22, 13; HRMS (FAB) calcd
for C.sub.16 H.sub.16 O.sub.3 256.1099, found 256.1099.
EXAMPLE 7
Compound 10
##STR16##
R.sub.f : 0.32 (petroleum ether:EtOAc=2:1); 49 percent yield; IR
(CCl.sub.4) .nu..sub.max 2927, 2343, 1680, 1660 cm.sup.-1 ; .sup.1 H NMR
(500 MHz, THF-d.sub.8) .delta. 2.85-2.82 (m, 2H, COCH.sub.2), 2.74-2.70
(m, 2H, C.ident.CCH.sub.2), 2.20-2.10 (m, 2H, --CH.sub.2 --), 2.01 (s, 6H,
2 aromatic CH.sub.3); MS (EI) 254 (M.sup.+ of the cyclized product upon
heating), 226, 198, 170, 149, 115.
EXAMPLE 8
Compound 11
##STR17##
R.sub.f : 0.20 (petroleum ether:EtOAc=4:1); 25 percent yield; IR
(CCl.sub.4) .nu..sub.max 3518, 2959, 2871, 1750 cm.sup.-1 ; .sup.1 H NMR
(500 MHz, DMSO-d.sub.6) .delta. 7.80, 7.75 (2s, 1H, H-9), 7.29 (s, 1H,
H-10), 5.35 (brs, 1H, OH), 4.66 (brs, 1H, CHOH), 2.91-2.80 (m, 2H, H-4),
2.14 (s, 6H, 2 aromatic CH.sub.3), 2.00-1.88 (m, 2H, H-2), 1.72-1.63 (m,
2H, H-3), 1.53, 1.52 (2s, 18H, 2.times.C(CH.sub.3).sub.3); HRMS (FAB)
calcd for C.sub.26 H.sub.34 O.sub.5 Cs (M+Cs) 559.1461, found 559.1472.
EXAMPLE 9
Compound 12
##STR18##
R.sub.f : 0.25 (petroleum ether:EtOAc=2:1); 40 percent yield; UV
(CHCl.sub.3) .lambda..sub.max (relative intensity) 226 (0.51), 262 (0.84),
343 (0.11) nm; IR (CCl.sub.4) .nu..sub.max 3610, 2931, 2874, 1720, 1660
cm.sup.-1 ; .sup.1 H NMR (500 MHz, CDCl.sub.3) .delta. 8.16, 7.79 (2s, 2H,
H-9, H-10), 4.86 (t, J=5.0 Hz, 1H, H-1), 2.95-2.80 (m, 2H, H-3), 2.12-1.96
(m, 2H, H-4), 1.95-1.78 (m, 2H, H-3); MS (EI) 256 (M.sup.+), 238, 228,
200, 128, 115; HRMS (EI) calcd for C.sub.16 H.sub.16 O.sub.3 256.1099,
found 256.1100.
EXAMPLE 10
Compound 13
##STR19##
R.sub.f : 0.19 (petroleum ether:EtOAc=4:1); 30 percent yield: IR
(CCl.sub.4) .nu..sub.max 1698, 1663, 1552 cm.sup.-1 ; .sup.1 H NMR (500
MHz, CDCl.sub.3) .delta. 8.70, 7.95 (2s, <2H, H-9, H-10), 3.11 (t, J=6.5
Hz, 2H, H-2), 2.74 (t, J=7.0 Hz, 2H, H-4), 2.21-2.15 (m, 8H, H-3 and
aromatic CH.sub.3); MS (EI) 56, 255, 254 (M.sup.+ +2, M.sup.+ +1,
M.sup.+), 228, 200, 172, 117; HRMS (EI) calcd for C.sub.16 H.sub.12
O.sub.3 D.sub.2 256.1068, found 256.1074.
EXAMPLE 11
DNA Cleavage Studies
To a vial containing 9 microliters of a cell-free .phi.X174 Type I
double-stranded DNA in pH 7.4 50 micromolar Tris-HCl buffer was added 1
microliter of a 1.0 millimolar or other appropriate ethanol solution of
Compounds 7, 8, 9, 10 and 12.
The vials were then placed in a 37.degree. C. oven for 15 hours. A 2.0
microliter portion of glycerol loading buffer solution containing
bromothymol blue indicator was added to each vial. A 10 microliter aliquot
was then drawn from each. Gel electrophoresis analysis of the aliquots was
performed using a 1.0 percent agarose gel with ethidium bromide run at 115
volts for one hour. DNA cleavage was indicated by the formation of Type II
DNA, which was detected by visual inspection of the gel under 310
nanometer ultraviolet light.
EXAMPLE 12
Screening Against Cancerous Cell Lines
In addition to the DNA cleavage screening already discussed, several of the
before-described compounds were screened against a panel of twelve
cancerous cell lines as target cells and four "normal" cell preparations.
This screening utilized a sulforhodamine B cytotoxicity assay as discussed
below.
SULFORHODAMINE B CYTOTOXICITY ASSAY
1. Preparation of target cells in 96-well plates
a. Drain media from T.sub.75 flask of target cell line(s) and carefully
wash cell monolayer two times with sterile PBS (approximately 5 mL per
wash)
b. Add 5 mL trypsin/EDTA solution and wash monolayer for approximately 15
seconds
c. Drain all but approximately 1 mL of trypsin/EDTA from flask, cap flask
tightly, and incubate at 37.degree. C. for approximately two to five
minutes until cells come loose.
d. Add 10-15 mL tissue culture (T.C.) medium (RPMI 1640 plus 10 percent
fetal calf serum and 2 mM L-glutathione) to flask and pipet gently up and
down to wash cells.
e. Remove a 1/2 mL aliquot of the cell suspension and transfer to a glass
12.times.75 mm culture tube for counting.
f. Count cells on a hemacytometer using trypan blue, and determine percent
viability.
g. Adjust volume of cell suspension with T.C. media to give a density of
1.times.10.sup.5 cells/mL.
h. Add 100 .mu.L of T.C. medium to wells A1 and B1 of a 96-well plate for
blanks.
i. Add 100 .mu.L of cell suspension to the remaining wells of the 96-well
plates.
j. Incubate plates for 24 hours at 37.degree. C., 5-10 percent CO.sub.2 in
a humidified incubator.
2. Preparation of sample drugs and toxic control
a. Stock drug solutions were prepared by dissolving drug in the appropriate
solvent (determined during chemical characterization studies) and sterile
filtering the drug-solvent solution through a sterile 0.2.mu. filter unit.
An aliquot was taken from each filtered drug solution and the O.D. was
measured to determine the drug concentration.
b. Dilute the stock drug solution prepared above with T.C. medium to the
desired initial concentration (10.sup.-2 -10.sup.-4 M). A minimum volume
of 220 .mu.L of diluted drug is required per 96-well plate used in the
assay.
c. Prepare toxic control by diluting stock doxorubicin solution to
10.sup.-7 to 10.sup.-9 M in T.C. medium. A minimum volume of 300 .mu.L is
required per 96-well plate.
3. Addition of Sample Drugs, Compounds and Controls to 96-well Plates
a. Remove and discard 100 .mu.L of T.C. medium from the wells in Column #2
of the 96-well plate using a multi-channel pipettor and sterile tips.
b. Add 100 .mu.L of the initial compound dilution to adjacent duplicate
wells in Columns #2. (Four materials can be tested in duplicate per
96-well plate.)
c. Remove 10 .mu.L of diluted compound from the wells in Column #2 and
transfer to the corresponding wells in Column #3. Mix by pipetting up and
down gently approximately five times.
d. Transfer 10 .mu.L to the appropriate wells in Column #4 and continue to
make 1:10 dilutions of compound across the plate through Column #12.
e. Remove and discard 100 .mu.L of medium from wells F1, G1, and H1. Add
100 .mu.L of toxic control (Doxorubicin diluted in T.C. medium) to each of
these wells.
f. Incubate (37.degree. C., 5-10 percent CO.sub.2 in humidified incubator)
plates for a total of 72 hours. Check plates at 24 hour intervals
microscopically for signs of cytotoxicity.
4. Cell Fixation
a. Adherent cell lines:
1. Fix cells by gently layering 25 .mu.L of cold (4.degree. C.) 50 percent
trichloroacetic acid (TCA) on top of the growth medium in each well to
produce a final TCA concentration of 10 percent.
2. Incubate plates at 4.degree. C. for one hour.
b. Suspension cell lines:
1. Allow cells to settle out of solution.
2. Fix cells by gently layering 25 .mu.L of cold (4.degree. C.) 80 percent
TCA on top of the growth medium in each well.
3. Allow cultures to sit undisturbed for five minutes.
4. Place cultures in 4.degree. C. refrigerator for one hour.
c. Wash all plates five times with tap water.
d. Air dry plates.
5. Staining Cells
a. Add 100 .mu.L of 0.4 percent (wt./vol.) Sulforhodamine B (SRB) dissolved
in 1 percent acetic acid to each well of 96-well plates using multichannel
pipettor.
b. Incubate plates at room temperature for 30 minutes.
c. After the 30 minute incubation, shake plates to remove SRB solution.
d. Wash plates two times with tap water and 1.times. with 1 percent acetic
acid, shaking out the solution after each wash. Blot plates on clean dry
absorbent towels after last wash.
e. Air dry plates until no standing moisture is visible.
f. Add 100 .mu.L of 10mM unbuffered Tris base (ph 10.5) to each well of
96-well plates and incubate for five minutes on an orbital shaker.
g. Read plates on a microtiter plate reader at 540 nM.
IC.sub.50 values; i.e., the concentration of Compound required to kill
one-half of the treated cells, where then calculated.
The cell lines assayed are listed below along with their respective
sources:
______________________________________
Cell Type Cell Line
______________________________________
Human Mammary Epithelial Cells
HMEC
Normal Human Dermal Fibroblast
NHDF
Normal Human Epidermal Keratinocytes
NHEK
Chinese Hamster Ovary CHO
Cancer Cell Lines
Melanoma SK-Mel-28
Ovarian Carcinoma Ovcar-3
Cervical Carcinoma SIHA
Breast Carcinoma MCF-7
Renal Carcinoma 786-0
Lung Carcinoma H-322
Lung Carcinoma UCLA P-3
Colon Carcinoma HT-29
Pancreatic Carcinoma Capan-1
Mouse Leukemia P-388
Promyeocytic Leukemia HL-60
T-Cell Leukemia Molt-4
______________________________________
UCLA P3 cells were provided by Dr. R. Reisfeld of The Scripps Research
Institute, and were originally obtained from Dr. D. Morton, University of
California, Los Angeles. P3 is a human nonsmall cell lung carcinoma cell
line. HMEC, NHDF and NHEK cells were obtained from Clonetics Corporation,
San Diego, CA. All other cells or cell lines were obtained from the
American Type Culture Collection (ATCC) (all except CHO cells are human o
mouse cancer cell lines as described by the ATCC).
Separate control studies were also carried out using the following well
known anticancer drugs with the following IC.sub.50 values for NHDF and
cancer cells.
______________________________________
Range of Average IC.sub.50 Values (Molarity)
Drug NHDF Cancer Cells
______________________________________
Doxorubicin -- 1.6 .times. 10.sup.-10 -9.8 .times. 10.sup.-8.sup
.
Dynemicin A 10.sup.-8 1.6 .times. 10.sup.-8 -9.8 .times. 10.sup.-10
Calicheamicin 2.5 .times. 10.sup.-9
5 .times. 10.sup.-5 -10.sup.-12 *
Morpholinodoxorubicin
-- 1.6 .times. 10.sup.-7 -9.8 .times. 10.sup.-9.sup.
Taxol 10.sup.-8 10.sup.-7 -10.sup.-9
Methotrexate 5 .times. 10.sup.-5
>10.sup.-4 -10.sup.-8
Cis-Platin 5 .times. 10.sup.-5
10.sup.-4 -10.sup.-6
Melphelan 10.sup.-4 10.sup.-4 -10.sup.-6
______________________________________
* Molt4 cells were susceptible at 10.sup.-12 M. All other cells were
susceptible at 3.9 .times. 10.sup.-9 M or higher concentrations.
______________________________________
CYTOTOXICITY OF COMPOUNDS 7-10
AGAINST VARIOUS CELLS
IC.sub.50 Values for
Compound Numbers*
CELLS 7 9 8 10
______________________________________
Normal
NHDF 1e-4 2.5e-5 3.1e-6
1e-4
HMEC 1e-4 2.5e-5 3.1e-6
1e-4
NHEK 2.5e-5 1.3e-5 3.1e-6
1e-4
CHO 1e-4 1.3e-5 3.2e-6
1e-4
Cancerous
SK-Mel-28 1e-4 2.5e-5 6.3e-6
1e-4
CAPAN-1 1.3e-5 1.3e-5 3.1e-6
1e-4
H322 1e-4 2.5e-5 1.3e-5
1e-4
UCLA P-3 1.3e-5 2.5e-5 6.3e-6
5e-5
MCF-7 5e-5 2.5e-5 6.3e-6
1.3e-5
OVCAR-3 1.3e-5 2.5e-5 6.3e-6
2.5e-5
HT-29 1.3e-5 2.5e-5 6.3e-6
5e-5
SIHA -- -- -- 1.3e-5
786-0 -- -- -- 5e-5
HL-60 1e-4 1.6e-6 7.8e-6
2.5e-5
MOLT-4 1e-4 5e-5 1e-7
1.19e-6
p-388 1.3e-5 3.1e-6 2e-6
--
______________________________________
*IC.sub.50 values are expressed as molarity. The abbreviation "e" is use
for an exponent power of ten rather than a natural log. Thus "1e5" is "1
.times. 10.sup.-5 M", etc.
As is seen from the above data, Compounds 8 and 10 exhibited cytotoxicity
potencies against cancer cells similar to those of methotrexate,
cis-Platin and melphelan. Compound 8 was similarly toxic to the normal
NHDF and cancer cells except Molt-4 cells, whereas Compound 10 was less
toxic than those well known anticancer drugs against the NHDF cells.
Although the present invention has now been described in terms of certain
preferred embodiments, and exemplified with respect thereto, one skilled
in the art will readily appreciate that various modifications, changes,
omissions and substitutions may be made without departing from the spirit
thereof.
Top